The estrogen receptor (ER) is a ligand-activated protein that mediates the actions of estrogens and antiestrogens in target cells. ER interaction with estrogen response elements (EREs) in target genes is responsible for the diverse effects of estrogens in regulating proliferation and other properties of breast cancer cells and cells of the reproductive tract. Although the interaction of the ER with the ERE is of paramount importance in the activation of estrogen-responsive genes, the mechanism by which this interaction initiates changes in gene expression is not well understood. We will use protease sensitivity assays to examine the effects of ERE sequence on ER conformation and determine if estrogen or antiestrogen binding alters the conformation of the ERE-bound receptor. DNase I, methylation interference, and hydroxyradical footprinting will provide complementary information about protein-DNA contacts involved in the interaction of ER with the consensus vitellogenin A2 ERE or the imperfect pS2 ERE. We will determine if DNA-induced changes in ER conformation are responsible for association of the ERE-bound receptor with selected proteins and whether the binding of these proteins could exert an additional level of control in the regulation of estrogen- responsive genes. Transcription factors associated with the A2 and pS2 EREs will be isolated and those which uniquely associate with either the A2 or the pS2 ERE-bound ER will be identified. These studies will provide new insights into estrogen regulation of gene transcription and a better understanding of how the ER functions in breast cancer and normal reproductive target cells.